1. Field of the Disclosure
The present invention relates generally to methods and compositions for measuring the amount of vitamin D derivatives. More particularly, the invention relates to the use of Fluorescence Resonance Energy Transfer (FRET) and a modified ligand-binding domain of the vitamin D receptor (LBD-VDR) to measure vitamin D derivatives.
2. Brief Description of Related Technology
Cholecalciferol and ergocalciferol (collectively are referred to as “Vitamin D”) are fat-soluble seco-steroid precursors to Vitamin D prohormones. The Vitamin D metabolites known as 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3 (collectively referred to herein as “25-hydroxyvitamin D”) are fat-soluble steroid prohormones to Vitamin D hormones that contribute to the maintenance of normal levels of calcium and phosphorus in the bloodstream. Cholecalciferol and ergocalciferol are normally present at stable, low concentrations in human blood. Both cholecalciferol and ergocalciferol are metabolized into prohormones by enzymes primarily located in the liver of the human body. Cholecalciferol is metabolized into a prohormone 25-hydroxyvitamin D3, and ergocalciferol is metabolized into two prohormones, 25-hydroxyvitamin D2 and 24(S)-hydroxyvitamin D2.
The Vitamin D prohormones are further metabolized in the kidneys into potent hormones. The prohormone 25-hydroxyvitamin D3 is metabolized into a hormone 1α,25-dihydroxyvitamin D3 (or calcitriol); likewise, 25-hydroxyvitamin D2 and 24(S)-hydroxyvitamin D2 are metabolized into hormones known as 1α,25-dihydroxyvitamin D2 and 1α,24(S)-dihydroxyvitamin D2, respectively. Production of these hormones from the prohormones also can occur outside of the kidney in cells which contain the required enzyme(s).
Surges in blood or intracellular prohormone concentrations can promote excessive extrarenal hormone production, leading to local adverse effects on calcium and phosphorus metabolism. Such surges also can inhibit hepatic prohormone production from subsequent supplemental Vitamin D and promote catabolism of both Vitamin D and 25-hydroxyvitamin D in the kidney and other tissues.
The Vitamin D hormones have essential roles in human health which are mediated by intracellular Vitamin D receptors (VDR). In particular, the Vitamin D hormones regulate blood calcium levels by controlling the absorption of dietary calcium by the small intestine and the reabsorption of calcium by the kidneys. Excessive hormone levels can lead to abnormally elevated urine calcium (hypercalciuria), blood calcium (hypercalcemia) and blood phosphorus (hyperphosphatemia). The Vitamin D hormones also participate in the regulation of cellular differentiation and growth, parathyroid hormone (PTH) secretion by the parathyroid glands, and normal bone formation and metabolism. Further, Vitamin D hormones are required for the normal functioning of the musculoskeletal, immune and renin-angiotensin systems. Numerous other roles for Vitamin D hormones are being postulated and elucidated based on the documented presence of intracellular VDR in nearly every human tissue.
The actions of Vitamin D hormones on specific tissues depend on the degree to which they bind to (or occupy) the intracellular VDR in those tissues. Cholecalciferol and ergocalciferol have affinities for the VDR which are estimated to be at least 100-fold lower than those of the Vitamin D hormones. As a consequence, physiological concentrations of cholecalciferol and ergocalciferol exert little, if any, biological actions without prior metabolism to Vitamin D hormones. However, supraphysiologic levels of cholecalciferol and ergocalciferol, in the range of 10 to 1,000 fold higher than normal, can sufficiently occupy the VDR and exert actions like the Vitamin D hormones. Similarly, the prohormones 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3 have essentially identical affinities for the VDR which are also estimated to be at least 100-fold lower than those of the Vitamin D hormones. As a consequence, physiological concentrations of 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3 have little, if any, biological actions without prior metabolism to Vitamin D hormones. However, supraphysiologic levels of 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3, in the range of 10 to 1,000 fold higher than normal, can sufficiently occupy the VDR to exert actions like the Vitamin D hormones.
As with most nuclear receptors, VDR undergoes a conformational change upon ligand binding (helix 12 folds underneath H4; Rochel, N., et al., Mol. Cell. 5, 173-179 (2000); Nayeri, S and Carlberg, C. Biochem J. 327, 561-568 (1997)) The ligand binding domain of human VDR is comprised of amino acids ˜118-427. Amino acid residues involved in hydrogen bonding to the ligand include Ser-237, Arg-274, Tyr-143, Ser-278, His-305 and His-397. Amino acids that interact with the ligand through non-hydrogen bonding interactions include Tyr-147, Phe-150, Leu-227, Leu-230, Leu-233, Val-234, Ile-271, Ser-275, Trp-286, Cys-288, Val-300, Leu-309, Leu-313 and Val-418. Val-418 is located on the activation helix (helix 12) and is likely to undergo change in proximity as a result of ligand-induced conformational changes (Rochel, N., et al., Mol. Cell. 5, 173-179 (2000)).
One method that may be used to monitor protein-protein interactions is Fluorescence Resonance Energy Transfer (FRET) (Berrera et al., Handb. Exp. Pharmacol. 186, 285-298 (2008)). FRET microscopy detects energy transfer from a higher-energy donor fluorochrome to a lower-energy acceptor fluorochrome when they are close together. Resonance energy transfer is a mechanism by which energy is transferred directly from one molecule to another. This only occurs over a very small distance, usually less than 10 nm, which is on the order of the size of a typical protein. When each member of a protein-protein pair is labeled with appropriate fluorophores (donor and acceptor), FRET can be used to detect when the proteins are in proximity. It may also be possible to use FRET to detect conformational changes in a single protein tagged with two fluorophores.